Purification and characterization of chitinases from Paecilomyces variotii DG-3 parasitizing on Meloidogyne incognita eggs.
Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60 degrees C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag(+) and Hg(2+) while Chi46 by Hg(2+) and Pb(2+) at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co(2+). On analyzing the hydrolyzates of chitin oligomers [(GlcNAc)( n ), n = 2-6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.
Nguyen
Van-Nam
VN
Division of Applied Bioscience and Biotechnology, Institute of Agricultural Science and Technology, Chonnam National University, Gwangju, 500-757, South Korea. ngdaonam963@yahoo.com
Oh
In-Jae
IJ
Kim
Young-Ju
YJ
Kim
Kil-Yong
KY
Kim
Young-Cheol
YC
Park
Ro-Dong
RD
D000595:Amino Acid Sequence; D000818:Animals; D001709:Biotechnology; D002688:Chitinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006790:Host-Parasite Interactions; D006999:Hypocreales / Q000145:classification* / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010063:Ovum / Q000382:microbiology*; D014431:Tylenchoidea / Q000254:growth & development / Q000382:microbiology*
D002688:Chitinases